The long range goal of these studies is to understand the molecular organization, diversity and regulation of tight junctions of the gastrointestinal tract and liver. The tight junction forms the molecular seal limiting movement of water and solutes between epithelial cells thus is a fundamental determinant of transport properties. Dynamic changes in its sealing properties occur during development, during crypt to villus differentiation of enterocytes, and in response to physiologic stimuli and hormones. A breakdown in junction integrity contributes to numerous pathologic processes including some forms of hepatic cholestasis and diarrheal disease. There is evidence that the tight junction also serves as a barrier to maintain the separation of biochemically distinct apical and basolateral membrane domains. Despite its obvious importance to epithelial function in the liver and gut, molecular characterization of the junction has been impossible until the identification of its first protein component, ZO-1. ZO-1 appears to be a very important component of the junction since it is present in all junction, is tightly associated with the cytoplasmic surface of membrane contacts and there is a correlation between ZO-1 isoforms and junction morphology. ZO-1 is not abundant and has proven difficult to isolate and study thus, the proposed approaches rely on molecular biologic techniques using recombinant fusion proteins and cultured epithelial cell transfection studies to circumvent these limitation. The specific goals are to increase basic knowledge of ZO-1 by sequencing its full length human cDNA. The proteins structural/functional organization will be defined by seeking membrane binding activity is fusion proteins as well as transfected sequences. We will test the hypothesis that an intact junction is required to maintain cell polarity by disrupting the junction in cultured cells with transfected ZO-1 sequences. Finally we will investigate the hypothesis that ZO-1 isoforms, generated by alternate RNA splicing, contribute to junction diversity throughout the GI tract and are altered in extrahepatic cholestasis which is accompanied by junction disruption.